Attenuated polioviruses

ABSTRACT

An attenuated enterovirus or rhinovirus, suitable for use as a vaccine, has a reversed base pairing in the part, or in a part corresponding to the part, of the 5&#39; non-coding region of the genome of poliovirus type 3 Leon strain shown below: ##STR1## A suitable attenuated poliovirus has the bases G and C at positions 469 and 534 respectively for a type 1 or type 2 poliovirus or at positions 472 and 537 respectively for a type 3 poliovirus.

This is a continuation of application Ser. No. 07/721,442, filed Jul.16, 1991, now abandoned.

This invention relates to the construction of vaccines againstrhinoviruses and enteroviruses, particularly polioviruses, by theintroduction of defined mutations into their genomes. These mutationsattenuate the virulence of wild type viruses and can further attenuateexisting live attenuated vaccine virus strains, thereby making them lesslikely to revert to virulence.

At the present time, the only vaccines routinely used againstenterovirus and rhinovirus infections are those against poliomyelitis.Of these the live attenuated vaccines developed by Sabin in the 1950'shave found greatest use throughout the world. Vaccine strains-derivedfrom each of the three poliovirus serotypes (P1, P2 and P3) wereprepared by passage of wild type viruses in cell cultures and wholeanimals until attenuated strains were obtained. These attenuated virusesare substantially less able to cause poliomyelitis in humans than theoriginal wild type strains. They are administered orally and replicatein the gut to induce a protective immune response.

Although these vaccines are generally regarded as safe, their use isassociated with a low level of paralysis in vaccinees. This is mostoften associated with type 2 and type 3 serotypes and rarely, if ever,with type 1. There is therefore a requirement for improved type 2 andtype 3 vaccines which would be comparable in safety to the excellenttype 1 strain. There is also a requirement for vaccines against otherenteroviruses, e.g. echo, coxsackie and hepatitis A, and againstrhinoviruses.

The Sabin vaccine strains were developed by essentially empiricalprocedures. The genetic basis of their attenuation is not properlyunderstood. Over the past few years, however, scientists have employed anumber of molecular biological techniques in an attempt to elucidate themechanism by which the neurovirulence of these vaccine strains isreduced. Most of the work has concentrated on serotypes 1 and 3. Forboth of these the complete nucleotide sequences of the vaccine strainshave been compared with those of their neurovirulent progenitors.

In the case of poliovirus type 1, the vaccine strain differs from itsprogenitor at 47 positions in the 7441 base genome (Nomoto et al, 1982,Proc Natl Acad Sci USA 79: 5793-5797). All of these are simple pointmutations and 21 of them give rise to amino acid changes in virus codedproteins. Although several mutations are thought to contribute to theattenuation phenotype of the vaccine strain, direct evidence has beenpresented that the mutation of A to G at position 480 in the 5'non-coding region of the genome has a marked attenuating effect on thevirus (Nomoto et al, 1987, UCLA Symp Mol Cell Biol, New Series, Vol 54(Eds M. A. Brinton and R. R. Rueckert), 437-452, New York: Alan R. LissInc).

Analogous studies on poliovirus type 3 reveal just 10 nucleotidesequence differences in the 7432 base genome between the vaccine and itsprogenitor strain (Stanway et al, 1984, Proc Natl Acad Sci USA 81:1539-1543). Just three of these give rise to amino acid substitutions invirus encoded proteins. The construction of defined recombinants betweenthe vaccine and its progenitor strain has allowed the identification ofthe mutations which contribute to the attenuation phenotype. One ofthese is at position 2034 and causes a serine to phenylalanine change invirus protein VP3.

The other mutation of interest is C to U at position 472 in the 5'non-coding region of the genome. This latter mutation has been observedto revert to the wild type C rapidly upon replication of the virus inthe human gut (Evans et al, 1985, Nature 314: 548-550). This reversionis associated with an increase in neurovirulence. C at position 472 hasalso been shown to be essential for growth of a mouse/human poliorecombinant virus in the mouse brain (La Monica et al, 1986, J Virol 57:515-525). Recently, we have observed that at 481 in poliovirus type 2 Achanges to G in an analogous fashion upon replication of the type 2vaccine in the gut of vaccinees.

In EP-A-0323900 attenuated enteroviruses in particular polioviruses, andrhinoviruses are described which have an attenuating mutation at aposition which is, or corresponds with, position 479 or 482 ofpoliovirus type 3 Leon strain. The attenuated viruses may also have anattenuating mutation at position 472.

A new model for the secondary structure of the 5' non-coding RNA ofpoliovirus type 3 Leon strain has now been proposed by us (Skinner etal, 1989, J. Mol. Biol, 207, 379-392). In the model, bases in the region470 to 540 are paired as follows: ##STR2##

We have found that a poliovirus with the base pair 472-537 reversed,i.e. 472 G and 537 C, is attenuated. Further, this attenuated virus hasa slightly lower LD₅₀ value than the corresponding poliovirus which onlyhas the mutation C to G at position- 472 but which retains the wild-typeG at position 537. There is no selective pressure on a poliovirus tomutate to a more attenuated poliovirus, so the attenuation in the doublemutant poliovirus may be locked in.

The findings can be extrapolated to all polioviruses. Indeed, they maybe extrapolated to other enteroviruses and rhinoviruses. Mutationsresulting in the reversal of a base pair at sites of other enterovirusesand rhinoviruses corresponding to the positions shown above forpoliovirus type 3 Leon strain can lead to attenuation. There is arelatively high degree of homology between the genome RNA of allenteroviruses and rhinoviruses. The positions of another strain ofenterovirus or rhinovirus corresponding to the positions of poliovirustype 3 Leon strain (based on the numbering used in the Stanway et alpaper already referred to) can be determined by lining up the sequencesof the genomic RNA of the strains.

Accordingly the invention relates to attenuated enteroviruses andrhinoviruses in which there is a reversal of a base pairing in the part,or in a part corresponding to the part, of the 5' non-coding region ofthe genome of poliovirus type 3 Leon strain shown below: ##STR3##

The accompanying FIGURE shows the relevant part of the 5' non-codingregion of poliovirus type 3 Leon strain in more detail. In accordancewith the present invention, one or more of the base pairs 471/538,472/537, 473/536, 477/534, 478/533, 479/532, 480/531, 481/530, 482,/529and 483/528 or of the corresponding base pairs of rhinoviruses or ofother enteroviruses may be reversed.

The present invention is particularly applicable to polioviruses. Theinvention is typically applied to a virus responsible for a disease orillness in humans. An attenuated poliovirus may be a type 1, type 2 ortype 3 poliovirus, for example a Sabin strain with a reversed basepairing according to the invention. Types 2 and 3 are preferred. Fortypes 1 and 2, positions 468, 474, 480, 525, 531 and 535 correspond topositions 471, 477, 483, 528, 534 and 538 respectively of poliovirustype 3.

Any base pair may be reversed to obtain an attenuated virus. More thanone base pair, for example 2 or 3 base pairs, may be reversed. We havefound in particular that the double mutation C to G at position 472 andG to C at position 537 in the 5' non-coding region of poliovirus type 3Leon strain causes attenuation. A useful attenuated poliovirus thereforehas the bases G and C at positions 472 and 537 respectively in the caseof type 3 polioviruses or at positions 469 and 534 respectively in thecase of type 1 or 2 polioviruses. Indeed a useful attenuated enterovirusor rhinovirus generally may have a reversed base pairing at the pairedpositions corresponding to positions 472 and 537 of poliovirus type 3Leon strain.

In a separate aspect of the invention, there are provided attenuatedenteroviruses, in particular polioviruses, and rhinoviruses having anattenuating mutation at least at a position which is, or correspondswith, position 537 of the genome of poliovirus type 3 Leon strain.Optionally there is also an attenuating mutation at position 472.

Such an attenuated poliovirus may be a type 1, type 2 or type 3poliovirus, for example a Sabin strain. Types 2 and 3 are preferred. Fortypes 1 and 2, positions 469 and 534 correspond to positions 472 and 537of poliovirus type 3 Leon strain. A useful attenuated poliovirustherefore has the base C at position 537 in the case of type 3polioviruses or at position 534 in the case of types 1 and 2polioviruses. Useful attenuated viruses with mutations at both positions472 and 537 or at the corresponding positions are as described above.

An attenuated virus according to the invention is prepared by a processcomprising:

(i) introducing the or each desired mutation by site-directedmutagenesis into a sub-cloned region, which includes the or eachposition it is wished to mutate, of a cDNA of an enterovirus orrhinovirus;

(ii) reintroducing the thus modified region into a complete cDNA fromwhich the region was derived; and

(iii) obtaining live virus from the cDNA thus obtained.

A mutation can be introduced into a strain of an enterovirus orrhinovirus, for example wild-type virus, by site-directed mutagenesis ofits genomic RNA. This may be achieved beginning with sub-cloning theappropriate region from an infectious DNA copy of the genome of any ofthe virus strain, for example a vaccine strain or its progenitor, intothe single strand DNA of a bacteriophage such as M13. The virus strainmay be a neurovirulent strain but is preferably a vaccine strain. Forpoliovirus it may be a Sabin, type 3 Leon or type 1 Mahoney strain. Theor each desired mutation is then introduced into this sub-cloned cDNAusing the technique of oligonucleotide directed mutagenesis.

After the introduction of mutations, the modified sub-cloned cDNAs arereintroduced into the complete cDNA from which they were derived and,for virulence testing in mice, into a cDNA derived from a murinepoliovirus derivative known to cause a poliomyelitis type disease inmice (La Monica et al, 1986). Live virus is recovered from the mutatedfull length cDNA by production of a positive sense RNA typically using aT7 promoter to direct transcription in vitro (Van der Werf et al, 1986,Proc Natl Acad Sci, USA 83: 2330-2334). The recovered RNA may be appliedto tissue cultures using standard techniques (Koch, 1973, Curr TopMicrobial Immunol 61: 89-138). After 4-6 days incubation virus can berecovered from the supernatant of the tissue culture. The level ofneurovirulence of the modified virus may then be compared with that ofthe unmodified virus using a standard LD50 test in mice (La Monica etal, 1986) or the WHO approved vaccine safety test in monkeys (WHO TechRep Ser 687: 107-175, 1983).

The attenuated viruses can be used as vaccines. They may therefore beformulated as pharmaceutical compositions further comprising apharmaceutically acceptable carrier or diluent. Any carrier or diluentconventionally used in vaccine preparations may be employed. Forexample, the presently used live attenuated poliovirus strains arestabilised in a solution of 1 molar MgCl₂ and administered as a mixtureof the three serotypes.

The attenuated viruses can therefore be used to prevent an infectionattributable to an enterovirus or rhinovirus in a human patient. Forthis purpose, they may be administered orally, as a nasal spray, orparenterally, for example by subcutaneous or intramuscular injection. Adose corresponding to the amount administered for a conventional livevirus vaccine, such as up to 10⁶ TCID₅₀ for a Sabin vaccine strain inthe case of poliovirus, may be administered.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows the base pairing in the relevant portion of the 5'non-coding of the poliovirus type 3 Leon strain.

The following Examples illustrate the invention.

EXAMPLE 1 Construction of In Vitro Mutants

A synthetic T7 polymerase promoter was cloned into pBR322 in anEcoRI-Hind3 linker to give pBRT7. The linker included a Stul site 5' ofthe Hind3 site. The sequence from the T7 polymerase promoter to the Stulsite of the resulting construct was:

    TTC GAA ATT AAT ACG ACT CAC TAT AGG CCT.

This enabled sequences to be ligated to the T7 promoter so that only 2extra Gs would be added to the 5' end of RNA transcripts. The extreme 5'end of pOLIO LEON (Stanway et al, 1984), which has a poly G tail and aPstl site as a result of the cDNA cloning procedure, was replaced by alinker of the sequence:

    TAT GAC GCG TGC GGC CGC AAG CTT TAA AAC.sub.7 . . . (polio 5' non-coding region) . . . GGTAC.sub.70.

The 5' end was therefore limited by a Hind3 site, whilst the Pstl siteand the poly G tail were removed. A Kpnl site, at position 70 in Leon,was used as the 3' end of the linker. Using this construction, the 5'end of pOLIO LEON from the Hind3 to a BamHI site (674), was cloned intopBRT7 to give pBRT75'L. The 5' non-coding region was brought under thecontrol of the T7 promoter by digesting pBRT75'L with Stul and Hind3,making the Hind3 site blunt-ended using mung bean nuclease andreligating to give p1-5 MBN. The resulting T7 promoter and 5' poliovirussequence thus read:

    TTCGAAATTAATACGACTCACTATAGGTTAAAC--.

An Sst I site at the 3' end of the hybrid poliovirus type 3 Leon/type 2lansing sequence in pVN23 (La Monica et al, 1986) was removed by partialSst I digestion, filling in by T4 polymerase and religation. Poliovirussequences from this plasmid were then cloned into p1-5 MBN, followingdigestion of both plasmids with Mlul and Sall, to give pT7SFP.Infectious virus could be recovered by linearising pT7SFP with Sall,synthesis of full length RNA using T7 polymerase and transfection ofHela cells with the RNA using the DEAE dextran method (Van der Werf etal, 1986).

The template for in vitro mutagenesis was made by subcloning the 5'non-coding region of poliovirus type 3 Leon from pVN23 (La Monica et al,1986) into the M13 derivative mICE 19 (Epernon, Nucl.Acids Res. 14,2830, 1986), using the Hind3 and Sst1 sites. A mutation was thenintroduced into this subcloned DNA fragment using the technique ofoligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids Res.10, 6487-6500, 1982). The oligonucleotide used for construct MAS 27-7was: CCA TGG TTA GCA TTA GCC GC.

Mutant 27-7 was constructed by hybridising this oligonucleotide to thesingle stranded cloned DNA fragment in the M13 phage derivative. Theoligonucleotide is complementary to the target region except at theposition to be mutated, where the base complementary to the desiredmutation is incorporated. The hybridised M13 and oligonucleotide DNAwere incubated in a reaction mixture containing DNA precursors and theenzymes DNA polymerase and DNA ligase. After incubation for one hour at37° C., closed circular DNA was isolated from this mixture by agarosegel electrophoresis. This DNA was then used to transform E coli mutS ormutL (deficient in DNA mismatch repair) which were then plated out on alawn of E coli JM101.

M13 plaques which arose on this lawn of E coli were picked andpropagated and single stranded M13 phage DNA isolated. The DNAs werethen sequenced using the method of Sanger and those with the desiredmutation were identified. From these, batches of replicative form doublestranded DNA were prepared and the Mlu1-Sst1 fragment containing 471base pairs of infectious poliovirus cDNA, which incorporates themutation, was recovered.

The mutated cDNA fragment was then reintroduced into pT7SFP. Live viruswas recovered from the mutated full length cDNA by the production of apositive sense RNA transcript from the T7 promoter in vitro (Van derWerf et al, 1986) which was applied to Hela cells in tissue cultureusing standard techniques (Koch, 1973). After 4 to 6 days incubation acytopathic effect was observed and virus could be recovered from thesupernatant.

Recovered mutant virus 27-7 with a G at position 472 was plaque purifiedand propagated in Hela cells. This virus pool was used for thepreparation of RNA on which the sequence of the virus mutant wasverified using primer extension nucleotide sequencing (Evans et al,1985). A portion of the pool was also used to assay neurovirulence.

ASSESSMENT OF VIRULENCE FOR MICE

Mutant virus 27-7 was grown in Hep2c cells, purified on sucrosegradients (Evans et al, 1985) and pelleted twice through phosphatebuffered saline (PBS) before resuspension in PBA at 10⁹ pfu per ml. C57black mice (obtained from OLAC), aged 21-28 days, were inoculatedintracranially with 10-20 μl of virus. Virus dilutions examined werefrom 10¹ to 10⁷ pfu per mouse in tenfold steps, and five or ten micewere used per dilution. Animals were observed once or twice a day for aperiod of up to 40 days post inoculation. The endpoint of the experimentfor the determination of the LD₅₀ value was set at 21 days. The LD₅₀value of mutant 27-7 was >10⁷ pfu.

Animals were sacrificed when full flaccid paralysis of one or more limbswas evident, and stored at -20° C. until the brain was removed for virusisolation. The brain was homogenised in 10 ml PBS, clarified by lowspeed centrifugation and filtered through a 0.22 μm filter beforeinoculation of 4 ml onto 10⁸ Hep2c cells. Five viruses were recovered.The recovered viruses were designated 27 plaque 1A, 27 plaque 2A, 27.7A,27.7B and 27.7C. Virus isolates were grown up, sequenced (Evans et al,1985) and reinoculated into mice to determine their LD₅₀ values.

The LD₅₀ values and sequences are shown in Table 1 below, together withthose for the original mutant 27-7 and for type 3 Leon. Differencesbetween the base sequence for type 3 Leon and that for the other virusesare underlined.

                  TABLE 1                                                         ______________________________________                                        Virus     LD.sub.50 (pfu)                                                                         Sequence (470-475 . . . 535-539)                          ______________________________________                                        Leon      10.sup.1  A U C C U A . . . C G G A A                               27-7      >10.sup.7 A U  .sub.-- G C U A . . . C G G A A                      27 plaque 1A                                                                            4 × 10.sup.5                                                                      A U  .sub.-- G C U A . . . C G G A A                      27 plaque 2A                                                                            10.sup.3                                                            27-7A     10.sup.2  A U C C U A . . . C G G A A                               27-7B     10.sup.1  A U C C U A . . . C G G A A                               27-7C     10.sup.6  A U  .sub.-- G C U A . . . C G  .sub.-- C A               ______________________________________                                                            A                                                     

These results show that mutant 27-7 is not a stable attenuated virusbecause viruses 27-7A and 27-7B, derived from 27-7, have back-mutated atposition 472 to become neurovirulent viruses having LD₅₀ values in theregion of 10¹ -10² pfu. However, virus 27-7C has an LD₅₀ value of 10⁶pfu and shows a compensating stabilising mutation at position 537. Ascan be seen from the accompanying FIGURE, bases 472 and 537 are paired.Virus 27-7C was deposited at the European Collection of Animal CellCultures, Porton Down, GB on 16 Jan. 1990 under accession number ECACC90011604.

EXAMPLE 2

In an attempt to identify more 27.7C type viruses, parental virus 27.7was again intracranially injected into mice. Viruses were recovered fromanimals displaying paralysis. The results are shown in Tale 2.

                  TABLE 2                                                         ______________________________________                                        Virus    Base at position:                                                    MAS 27.7 472                     537                                          ______________________________________                                        27.7 pl A                                                                              G                                 G                                  27.7 A   C                                 G                                  27.7 B   C                                 G                                  27.7 C   G                                 C                                  27.7 p 2A                                                                              C                                 G                                  27.7 p 2A/1A                                                                           C                                 G                                  27.7 p 2A/2A                                                                           C                                 G                                  27.7 p 2A/3A                                                                           C                                 G                                  27.7 p 2A/4A                                                                           C                                 G                                  27.7 p 2A/4B                                                                           C                                 G                                  27.7 D   C       T       T     A     A     G                                                   489     492   494   498                                      27.7 E   C                                 G                                  27.7 F   C                                 G                                  ______________________________________                                    

One 27.7C type virus was isolated from a group of 13 such recoveredviruses. In a further attempt to identify more 27.7C type viruses, virus27 plaque 1A (same sequence of 5' non-coding region as the parental 27.7virus) was injected intracranially into mice. Viruses were recoveredfrom animals displaying paralysis. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                       Base at position:                                              Virus            472     537                                                  ______________________________________                                        27 p1 1A         G       G                                                    27 p1 1A/1C      C       G                                                    27 p1 1A/1B      C       G                                                    27 p1 1A/2B      G       C                                                    27 p1 1A/2C      C       G                                                    27 p1 1A/3A      G       C                                                    27 p1 1A/3B      C       G                                                    27 p1 1A/4A      C       G                                                    27 p1 1A/5B      G       C                                                    27 p1 1A/6A      C       G                                                    27 p1 1A/7B      C       G                                                    27 p1 1A/7C      C       G                                                    ______________________________________                                    

A further three 27.7C type viruses were isolated from a group of 12recovered viruses. These 27.7C type viruses have been designated 27 pl1A/2B, 27 plaque 1A/3A and 27 pl 1A/5B.

It was hoped that when this virus 27.7C was passaged many times throughmice that the LD₅₀ value of this virus would be maintained at 10⁶ -10⁷p.f.u. Indeed, it is shown in Table 4 below that when 27.7C virus waspassaged for a further generation in mice (27.7C/2A-7D) that all 9recovered viruses from mice displaying paralysis were base pairedattenuating polioviruses (G at 472 and C at 537).

                  TABLE 4                                                         ______________________________________                                                       Base at position:                                              Virus            472      537                                                 ______________________________________                                        MAS 27.7         G        G                                                   27.7 C/2A        G        C                                                   27.7 C/2B        G        C                                                   27.7 C/4A        G        C                                                   27.7 C/5A        G        C                                                   27.7 C/5B        G        C                                                   27.7 C/6A        G        C                                                   27.7 C/7A        G        C                                                   27.7 C/7C        G        C                                                   27.7 C/7D        G        C                                                   27.7 C/2A/7A     G        C                                                   27.7 C/2A/7C     G        C                                                                    ALL G    ALL C                                               ______________________________________                                    

These 27.7C type viruses have now been passaged for a further threegenerations in mice. All polioviruses recovered from paralysed animalswere base paired attenuating (LD₅₀ >10⁶ p.f.u.) polioviruses (G at 472and C at 537). Some of our most recent data on the passage of these27.7C polioviruses in mice is summarised in Table 5 below.

                  TABLE 5                                                         ______________________________________                                                    Base at position:                                                 Virus         472       537    LD.sub.50                                      ______________________________________                                        27.7 Parental Virus                                                                         G         G      8.25 × 10.sup.6                          27.7C         G         C      2.63 × 10.sup.6                          (3 Viruses)                                                                   27.7C/2A-7D   G         C      2.57 × 10.sup.6                          (9 Viruses)                                                                   27.7C/2A/3A-7B                                                                              G         C      5.43 × 10.sup.6                          (4 Viruses)                                                                   ______________________________________                                    

EXAMPLE 3 Protective Immunity Elicited by 27.7C Type Virus

In a series of experiments groups of C57BL/6 mice were injectedintracranially with 10¹ -10⁷ p.f.u. of a 27.7C virus. Animals were thenobserved daily for paralysis up to Day 50. Surviving non-paralysed micewere then bled out and sera assayed for levels of neutralising antibodyto Lansing poliovirus. The results for three attenuated stable basepaired 27.7C type viruses are shown in Tables 6 to 8.

                  TABLE 6                                                         ______________________________________                                        Neutralising antibody elicited by 27.7C/2A/5B virus                                    Virus Titre-                                                                             Antibody Titre                                                                            Paralysed or                                  Day Number                                                                             Injected   to Lansing  Non-Paralysed                                 ______________________________________                                        25       10.sup.7   32          Non-Paralysed                                 25       10.sup.7   64          Non-Paralysed                                 26       10.sup.6   >256        Non-Paralysed                                 26       10.sup.6   16          Non-Paralysed                                 27       10.sup.5   128         Non-Paralysed                                 28       10.sup.5   16          Non-Paralysed                                 28       10.sup.5   <           Non-Paralysed                                 29       10.sup.5   <           Non-Paralysed                                 29       10.sup.5   8           Non-Paralysed                                 37       10.sup.4   <           Non-Paralysed                                 37       10.sup.4   <           Non-Paralysed                                 38       10.sup.4   <           Non-Paralysed                                 ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Neutralising Antibody Elicited by 27.7C/2A/7C Virus                                    Virus Titre-                                                                             Antibody Titre                                                                            Paralysed or                                  Day Number                                                                             Injected   to Lansing  Non-Paralysed                                 ______________________________________                                        27       10.sup.7   128         Non-Paralysed                                 30       10.sup.6    8          Non-Paralysed                                 31       10.sup.6   32          Non-Paralysed                                 32       10.sup.6   64          Non-Paralysed                                 32       10.sup.6   128         Non-Paralysed                                 33       10.sup.5   >256        Non-Paralysed                                 33       10.sup.5    8          Non-Paralysed                                 34       10.sup.5    2          Non-Paralysed                                 34       10.sup.5    4          Non-Paralysed                                 35       10.sup.4   <           Non-Paralysed                                 ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Neutralising Antibody Elicited by 27.7C/2A/7D Virus                                    Virus Titre-                                                                             Antibody Titre                                                                            Paralysed or                                  Day Number                                                                             Injected   to Lansing  Non-Paralysed                                 ______________________________________                                        39       10.sup.7   128         Non-Paralysed                                 39       10.sup.7   64          Non-Paralysed                                 40       10.sup.7   64          Non-Paralysed                                 40       10.sup.7   64          Non-Paralysed                                 41       10.sup.6   16          Non-Paralysed                                 41       10.sup.6   64          Non-Paralysed                                 43       10.sup.5   <           Non-Paralysed                                 43       10.sup.5   128         Non-Paralysed                                 44       10.sup.5    2          Non-Paralysed                                 45       10.sup.4   <           Non-Paralysed                                 45       10.sup.4   <           Non-Paralysed                                 46       10.sup.4   <           Non-Paralysed                                 ______________________________________                                    

EXAMPLE 4 Multiple Copies of 27.7C Virus Genome Obtained by PolymeraseChain Reaction (PCR)

p27.7C PCR was constructed using DNA obtained from the 27.7C virus usingpolymerase chain reaction. An oligonucleotide complimentary to thenucleotides 761-783 of poliovirus type 3 was used to reverse transcribeRNA extracted from purified 27.7C virus. A second oligonucleotidehomologous to nucleotides 36-60 was then used to amplify the region of27.7C between nucleotides 36-783 which contains the CG to GC mutationresponsible for the attenuation of revertant 27.7C. This PCR amplifiedDNA was digested with Mlul and Sst1 and cloned into pt7SFP (Skinner etal J. Mol. Biol. 207, 379-392, 1989), following digestion of the plasmidwith Mlu1 and Sstl, to give p27.7C PCR.

The p27.7C PCR sequence was checked between the Mlul and Sstl cloningsites by double stranded DNA sequencing. Infectious virus was recoveredby linearising p27.7C PCR with Sal1, synthesising full length RNA by T7polymerase and transfection of HeLa cells with RNA using the DEAEdextran method (van der Werf et al, 1986). The sequence of the recoveredvirus was determined through the inserted region, and the non-plaquepurified preparation had an LD₅₀ of greater than 10⁶ when tested inmice.

We claim:
 1. An attenuated poliovirus in which there is a reversal of abase pairing in the part, or in a part corresponding to the part, of the5' non-coding region of the genome of poliovirus type 3 Leon strainshown below: ##STR4## wherein the bases at positions 472 and 537 of thegenome of poliovirus type 3 Leon strain, or at positions correspondingto the said positions 472 and 537, are G and C respectively.
 2. Anattenuated virus according to claim 1, which is a type 1 poliovirus. 3.An attenuated virus according to claim 1, which is a type 2 poliovirus.4. An attenuated virus according to claim 1, which is a type 3poliovirus.
 5. A pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and an attenuated virusas claimed in claim
 1. 6. A method of vaccinating a patient against apoliovirus, which method comprises administering thereto an effectiveamount of a virus as claimed in claim
 1. 7. An attenuated poliovirushaving an attenuating mutation at least at a position which is, orcorresponds with, position 537 of the genome of poliovirus type 3 Leonstrain.